Bombesin- and CCK-related peptides are found widely in the gastrointestinal (GI) tract and central nervous system, however, aspects of their cellular basis of action remain unclear, particularly the role of tyrosine phosphorylation in their signaling cascade. In our studies the cellular basis of action of two bombesin (BN) receptors (neuromedin receptor [NMB-R], BRS-3 orphan receptor] and cholecystokinin-A (CCK) receptors [CCK-A-R] are being examined. BRS-3 is an orphan receptor that has a 60% homology to other mammalian BN receptors, however, its endogenous ligand, pharmacology and cell biology is almost completely unknown. To explore this, because no native cell lines exist two strategies were used involving stably overexpressing hBRS-3 receptors in a cell line possessing low numbers of native receptors and in a cell line in which other mammalian BN receptors when expressed function similar to native BN receptors. Stable cell lines were screened by Northern blotting and positive cell lines used to screen for a possible synthetic ligand by assessing changes in [3-H]IP. A novel ligand [DPhe-6, BetaAla-11,Phe-13,Nle-14]Bn(6-14) was identified which interacted with high affinity with BRS-3 receptors. This ligand was used to explore the cellular biology of BRS-3 receptors demonstrating they were coupled to phospholipase C (PLC), phospholipase D (PLD), and caused tyrosine phosphorylation of a number of proteins including p125-FAK and paxillin, but are not coupled to adenylate cyclase. In other studies using transfected cells containing NMB-R and native cells possessing this receptor, it was shown to be coupled to PLD, PLC and to cause tyrosine phosphorylation of p125-FAK in a PKC-independent manner which required the integrity of the actin cytoskeleton and the participation of the small GTP binding protein, p21-rho. Lastly, CCK-A-R activation was shown to be coupled to tyrosine phosphorylation of a number of novel intracellular proteins including p130-cas, p125-FAK, paxillin and PKC-delta. CCK caused translocation of these proteins to the plasma membrane and tyrosine phosphorylation of p130-cas resulted in its association with the important intracellular adapter protein, Crk.